D-serine transporter inhibitors as pharmaceutical compositions for the treatment of visual system disorders

ABSTRACT

The present invention relates to pharmaceutical compositions comprising D-serine transporter inhibitors and therapeutic methods using such pharmaceutical compositions in methods for the treatment of visual system disorders and the enhancement of the visual function.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional patent application of U.S.Non-Provisional patent application Ser. No. 13/479,803 filed May 24,2012, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/490,652 filed on May 27, 2011, which are incorporated here byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions comprisingD-serine transporter inhibitors and therapeutic methods using suchpharmaceutical compositions in methods for the treatment of visualsystem disorders and the enhancement of the visual function.

BACKGROUND

Neuronal circuits in the central nervous system rely on the release ofchemical neurotransmitters from specialized connections called synapsesfor communication. The major excitatory neurotransmitter is the aminoacid glutamate, and release of glutamate from a pre-synaptic terminalelicits a response through activation of several types of receptors. Oneof the sub-types of glutamate receptors, the N-methyl-D-aspartate (NMDA)receptor, plays a major role in neuronal communication and in theplasticity of synaptic responses that occurs under both physiologicaland pathophysiological conditions.

NMDA receptors are ligand-gated cation channels comprised of atetrameric assembly of NR1, NR2 and NR3 sub-units (Paoletti and Neyton,2007). They are unique amongst neurotransmitter receptors in that theyrequire occupation of two separate recognition sites for activation. Anacidic amino acid site where glutamate binds, is located on the NR2sub-units, and a neutral amino acid (or co-agonist) site is located onthe NR1 sub-unit. The endogenous co-agonist for this site was originallythought to be glycine, but more recent evidence indicated that D-serineis also an endogenous co-agonist. In fact, in higher brain regionsD-serine may be the dominant co-agonist. Occupation of the co-agonistsite is essential for glutamate (or a glutamate analog) to activate theNMDA receptor, and in native assays the removal of glycine or D-serineby exogenously-applied degradative enzymes can reduce or abolish NMDAreceptor-mediated responses. For example, in the rat hippocampal slice,application of the D-serine metabolizing enzyme, D-amino acid oxidase(D-AAO), completely prevents the induction of long-term potentiation(LTP) a form of synaptic plasticity whose initiation is dependent onNMDA receptor activation (Yang et al., 2003). This suggests that thedominant co-agonist in this case is D-serine, since glycine is not asubstrate for D-AAO.

The mechanisms that regulate extracellular D-serine, and thereforegovern how NMDA receptors are activated, are not well understood. Inkeeping with other neurotransmitters and neuromodulators, it is likelythat transporters on the cell surface are involved in regulatingsynaptic levels of D-serine. Amino acid transporters usually preferL-amino acids, however D-serine has been shown to be a substrate forcertain transporters. These include the heterodimeric transporter asc-1(SLC3A2/SLC7A10) which has micromolar affinity for D-serine, ASCT2(SLC1A5), ATB^(o+) (SLC7A9) and PAT1-4 (SLC36A1-4). Based on the tissueand cellular localization, the primary candidates for transporters thatregulate synaptic D-serine levels are asc-1 (neuronal) and ASCT2(glial). The related transporter ASCT1 (SLC1A4) also has been localizedto neurons and glia, however it has been reported that D-serine is not asubstrate for ASCT1 (Shafqat et al., 1993). None of these transportersare selective for D-serine, and their substrates are typically smallneutral amino acids such as serine, alanine, cysteine and threonine.They also are known to function as exchangers that can flux theirsubstrates both into and out of cells. Consequently, it has been unclearif these transporters are responsible primarily for the net uptake orthe net release of D-serine and other substrates. Considering that asc-1has the highest known affinity for D-serine, it has been thought thatthis transporter is primarily responsible for removing D-serine from theextracellular space (Rutter et al., 2007). In support of this, the asc-1knock-out mouse has a phenotype that includes increased excitability(Xie et al., 2005).

In the visual system, NMDA receptors are important mediators ofglutamate-mediated neurotransmission and synaptic plasticity. Thisoccurs at all levels of the visual axis, including neurons in theretina, in the central neurons that receive retinal ganglion cell inputin the lateral geniculate nucleus and the superior colliculus, and inthe visual cortex. Based on experiments using D-AAO, D-serine has beenshown to be an endogenous co-agonist involved in NMDA-receptor-mediatedsynaptic responses in the retina (Stevens et al., 2003) NMDA receptorshave also been shown to mediate synaptic responses in the lateralgeniculate (Harveit & Heggelund, 1990; Scharfman et al., 1990) and thevisual cortex (ie the primary pathways that transduce visualinformation). In the visual cortex, NMDA receptors mediate thephenomenon of long-term potentiation (LTP), an important form ofsynaptic plasticity. NMDA receptor-dependent LTP occurs in many brainregions and is viewed as a mechanism of synaptic strengthening that isfundamental to the establishment and maintenance of appropriate synapticconnections. In the hippocampus, for example, LTP has been studied as asynaptic surrogate of learning and memory. In visual cortex neurons, LTPmediates stimulus-specific response potentiation, a form ofexperience-dependent plasticity that contributes to visual function(Cooke and Bear, 2010).

In retinal diseases such as glaucoma and macular degeneration, loss ofthe vision arises from degeneration or malfunction of retinal cells.Consequently, the normal neuronal transmission along the visual pathwayis disrupted in the affected parts of the visual field. One strategy toremedy this loss of function would be to enhance the visualneurotransmission that remains unaffected by disease to compensate forthe region of impairment. In addition, enhancing the plasticity ofneuronal connections that occurs in the adult visual system could leadto the establishment of new neuronal connections that replace the lostfunction and improve visual performance.

Enhancing NMDA receptor activity by increasing the extracellular levelsof D-serine would boost visual performance and compensate for the lossof vision resulting from retinal disease. As a result, we havediscovered compounds that inhibit the transport of D-serine and enhanceNMDA receptor-mediated synaptic responses. We identified the D-serinetransporters that are important for regulating NMDA receptor-mediatedLTP in the visual cortex, and we demonstrated that D-serine transportinhibitors improve visual function in animal models of retinal disease.

SUMMARY OF THE INVENTION

The present invention relates to the use of pharmaceutical compositionscomprising D-serine transporter inhibitor compound(s) in methods for thetreatment of visual system disorders.

Visual system disorders which may be treated with the D-serine transportinhibitors include macular edema, dry and wet macular degeneration,choroidal neovascularization, diabetic retinopathy, acute macularneuroretinopathy, central serous chorioretinopathy, cystoid macularedema, and diabetic macular edema, uveitis, retinitis, choroiditis,acute multifocal placoid pigment epitheliopathy, Behcet's disease,birdshot retinochoroidopathy, syphilis, lyme, tuberculosis,toxoplasmosis, intermediate uveitis (pars planitis), multifocalchoroiditis, multiple evanescent white dot syndrome (mewds), ocularsarcoidosis, posterior scleritis, serpiginous choroiditis, subretinalfibrosis and uveitis syndrome, Vogt-Koyanagi- and Harada syndrome;retinal arterial occlusive disease, anterior uveitis, retinal veinocclusion, central retinal vein occlusion, disseminated intravascularcoagulopathy, branch retinal vein occlusion, hypertensive funduschanges, ocular ischemic syndrome, retinal arterial microaneurysms,Coat's disease, parafoveal telangiectasis, hemiretinal vein occlusion,papillophlebitis, central retinal artery occlusion, branch retinalartery occlusion, carotid artery disease (CAD), frosted branch angiitis,sickle cell retinopathy, angioid streaks, familial exudativevitreoretinopathy, and Eales disease; traumatic/surgical conditions suchas sympathetic ophthalmia, uveitic retinal disease, retinal detachment,trauma, photocoagulation, hypoperfusion during surgery, radiationretinopathy, and bone marrow transplant retinopathy; proliferativevitreal retinopathy and epiretinal membranes, and proliferative diabeticretinopathy; infectious disorders such as ocular histoplasmosis, oculartoxocariasis, presumed ocular histoplasmosis syndrome (POHS),endophthalmitis, toxoplasmosis, retinal diseases associated with HIVinfection, choroidal disease associate with HIV infection, uveiticdisease associate with HIV infection, viral retinitis, acute retinalnecrosis, progressive outer retinal necrosis, fungal retinal diseases,ocular syphilis, ocular tuberculosis, diffuse unilateral subacuteneuroretinitis, and myiasis; genetic disorders such as retinitispigmentosa, systemic disorders with associated retinal dystrophies,congenital stationary night blindness, cone dystrophies, Stargardt'sdisease and fundus flavimaculatus, Best's disease, pattern dystrophy ofthe retinal pigmented epithelium, X-linked retinoschisis, Sorsby'sfundus dystrophy, benign concentric maculopathy, Bietti's crystallinedystrophy, and pseudoxanthoma elasticum; retinal tears/holes such asretinal detachment, macular hole, and giant retinal tear; tumors such asretinal disease associated with tumors, congenital hypertrophy of theretinal pigmented epithelium, posterior uveal melanoma, choroidalhemangioma, choroidal osteoma, choroidal metastasis, combined hamartomaof the retina and retinal pigmented epithelium, retinoblastoma,vasoproliferative tumors of the ocular fundus, retinal astrocytoma, andintraocular lymphoid tumors; punctate inner choroidopathy, acuteposterior multifocal placoid pigment epitheliopathy, myopic retinaldegeneration, acute retinal pigement epitheliitis, retinitis pigmentosa,proliferative vitreal retinopathy (PVR), age-related maculardegeneration (ARMD), diabetic retinopathy, diabetic macular edema,retinal detachment, retinal tear, uveitus, cytomegalovirus retinitis,glaucoma, amblyopia, stroke-induced blindness, visual dysfunction inParkinson's disease, Alzheimer's disease and multiple sclerosis,seizure-induced cortical blindness, induced visual dysfunction, andepileptic blindness.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ability of selected amino acids to inhibit D-serinetransport into neurons (rat brain synaptosomes) and glia (rathippocampal astrocytes). Values are the concentration of amino acidrequired to inhibit 50% of [³H]D-serine transport (IC₅₀) in μM, and arethe means of at least two determinations. L-GPNA: L-γ-nitrophenylglutamyl anilide.

FIG. 1A represents a graph displaying the electrophysiological recordingfrom rat hippocampal slices and showing that L-4OHPG(L-4-hydroxyphenylglycine) potentiates NMDA receptor-mediated excitatorypostsynaptic currents (EPSC_(NMDA)). The representative experiment showsthat the potentiation effect of L-4OHPG (1000 μM) lasted for one hourand then returned to baseline in control buffer. 1000 is theconcentration of L-4OHPG in μM and 22% refers to the percentage increasein the amplitude of EPSC_(NMDA). Dots represent the amplitudes ofEPSC_(NMDA). Filled triangles represent control; open circles represent1 μM 7-CKY and filled circles represent 1000 μM L-4OHPG. 7-CKY is acompetitive inhibitor at the D-serine site of the NMDA receptor, whichinhibits the NMDA receptor-mediated EPSCs and increases the sensitivityof EPSC_(NMDA) to D-serine.

FIG. 1B represents a graph featuring summary data showing thedose-dependent effects of L-4OHPG on EPSC_(NMDA).

FIG. 2A represents a graph showing that L-4OHPG dose-dependentlyfacilitates long-term potentiation (LTP) in the primary visual cortex ofrats.

FIG. 2B represents a graph that features the results of exposing visualcortex slices to D-amino acid oxidase (DAAO), an enzyme that selectivelydegrades extracellular D-serine. The data suggest that L-4OHPG'senhancement of LTP in the visual cortex slice of rats is dependent onextracellular D-serine.

FIG. 3A represents a graph that shows the correlation between theability of compounds to inhibit neuronal D-serine transport and thethreshold concentration required to enhance LTP in the visual cortexslice. The r² value (correlation coefficient) and p value (probability)indicates that no significant correlation exists.

FIG. 3B represents a graph that shows the correlation between theability of compounds to inhibit astrocyte D-serine transport and thethreshold concentration required to enhance LTP in the visual cortexslice. The r² value (correlation coefficient) and p value (probability)indicates that a highly significant correlation exists.

FIG. 4 represents a graph that shows the 2-component inhibition ofD-serine transport into astrocytes by L-glutamine andL-trans-4-hydroxyproline (L-t-4OHPro) and IC₅₀ values for the individualcomponents. Two-component inhibition curves were fitted using analgorithm available in GraphPad Prism 4. “Component 1” is the highaffinity component and “Component 2” is the low affinity component.“Fraction” refers to the proportion that each component contributes tothe total inhibition.

FIG. 5 represents a graph that shows the inhibition of transport intoHEK cell lines expressing ASCT1 and ASCT2 by L-glutamine and L-t-4OHPro.

FIG. 6A represents a graph that shows transport of [³H]D-serine into HEKcells expressing ASCT1 or ASCT2. Transport was measured in the presence(control) and in the absence (zero Na) of extracellular sodium.

FIG. 6B represents a graph that shows the inhibition of [³H]L-serinetransport into astrocytes and HEK cells expressing ASCT1 and ASCT2 byD-serine.

FIG. 2 shows IC₅₀ values for the inhibition of [³H]L-serine transportinto astrocytes and HEK cells expressing recombinant human ASCT1 orASCT2. Values are IC₅₀'s in μM from 6-12 point inhibition curves, withan n of at least 2. For L-trans-4-hydroxyproline (L-t-4OHPro) andL-GPNA, two components of inhibition were present in the astrocyteassay, and *values are presented for the high and low affinitycomponents. For comparison, threshold concentrations for the enhancementof LTP in rat visual cortex slices are shown.

FIG. 7 represents the correlation of the IC₅₀ values from the transportassays (see Table 2 and accompanying explanation) with the LTP thresholddata (see FIG. 2A and accompanying explanation). Graph A shows thecorrelation between the IC₅₀ values for the ASCT1 transporter (alsoknown as SLC1A4) and the LTP threshold data; graph B shows thecorrelation between the IC₅₀ values for the ASCT2 transporter (alsoknown as SLC1A5) and the LTP threshold data; graph C shows thecorrelation between the product of both ASCT1's and ASCT2's IC₅₀ valuesand the LTP threshold data in order to take into account howcontributions from both transporters might be important to produce theLTP enhancement (this plot gives the best correlation and suggests thatinhibition of both transporters, leads to optimal LTP enhancement).

FIG. 8 shows that L-4OHPG enhances visual function in normal rats asassessed by sweep VEP. Saline was used as a control. Half of the ratswere injected with 30 mg/kg L-4OHPG and the other half with saline inthe first test; cross-over exposure took place one week later; thespatial frequencies were swept from 0.03 cycles per degree (cpd) to 1.6cpd. Graphs A and B show visual acuity (VA) values for each rat measuredbefore and 30 min after saline (A) or L-4OHPG (B) injection, and theaverage VA values from all rats are shown in D. The changes in VAmeasures are shown in C and E.

FIG. 9 shows that L-4-fluorophenylglycine (L-FPG) enhances visualfunction in normal rats as assessed by sweep VEP. Saline was used as acontrol. Half of the rats were injected with 10 mg/kg L-FPG and theother half with saline in the first test; cross-over exposure took placeone week later; the spatial frequencies were swept from 0.03 cpd to 1.6cpd. VA before and 40 min after L-FPG (graph A) or saline (graph B)injection are compared in individual rats and the average VA values areshown in D. The changes in VA measures are shown in C and E.

FIG. 10 shows the visual enhancement effect of L-4OHPG in the ONC rat.Saline was used as a control. Half of the rats were injected with 30mg/kg L-4OHPG and the other half with saline in the first test;cross-over exposure took place one week later; the spatial frequencieswere fixed at 0.2 cpd. Graph A and Graph B show data collected 30 minbefore and after L-4OHPG or saline injection. The changes in the powerof signals are shown in C and D.

FIG. 11 shows that L-4OHPG enhances visual function in normal rabbits asassessed by sweep VEP. Saline was used as a control. Half of the ratswere inject with 30 mg/kg L-4OHPG and the other half with saline in thefirst test; cross-over exposure took place one week later. the spatialfrequencies were swept from 0.03 cycles per degree (cpd) to 1.6 cpd.

FIG. 12 shows that 4-FPG improved the contrast sensitivity impaired inrats fifty-three weeks after blue-light treatment. Saline was used as acontrol. Half of the rats were injected with 10 mg/kg 4-FPG and theother half with saline in the first test; cross-over exposure took placeone week later; the spatial frequencies were fixed at 0.575 cpd.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect the invention relates to a method for the treatment ofvisual system disorders caused by a deficit in N-methyl-D-aspartatereceptor function, the method comprising administering to a subject inneed thereof an ophthalmically acceptable pharmaceutical compositioncontaining a therapeutically effective amount of one or more D-serinetransporter inhibitor compounds.

In another aspect the invention relates to a method for the treatment ofvisual system disorders, the method comprising administering to asubject in need thereof an ophthalmically acceptable pharmaceuticalcomposition containing a therapeutically effective amount of one or moreD-serine transporter inhibitor compounds selected from theGlycine/Alanine family, the Glutamine/Asparagine family, the TryptophanFamily, the Phenylglycine family, the Phenylalanine family, the Cysteinefamily, the Serine/Threonine family, the Cyclic Amino Acid family andthe Proline family.

In another aspect the invention relates to a method for the treatment ofvisual system disorders, the method comprising administering to asubject in need thereof an ophthalmically acceptable pharmaceuticalcomposition containing a therapeutically effective amount of one or moreD-serine transporter inhibitor compounds selected fromL-gamma-glutamyl-4-nitroanilide, L-4-hydroxyphenylglycine,L-4-fluorophenylglycine, L-phenylglycine, trans-4-hydroxy-L-proline andR-gamma-2,4-dichlorobenzyl-L-proline.

In another aspect the invention relates to a pharmaceutical compositioncomprising as active ingredient a therapeutically effective amount of atleast one D-serine transporter inhibitor compound and a pharmaceuticallyacceptable adjuvant, diluents or carrier.

In another aspect the invention relates to a method for the treatment ofvisual system disorders caused by a deficit in N-methyl-D-aspartatereceptor function, the method comprising administering to a subject inneed thereof an ophthalmically acceptable pharmaceutical compositioncontaining a therapeutically effective amount of at least one or moreASCT1 inhibitor compounds and/or at least one or more ASCT1 inhibitorcompounds.

In another aspect the invention relates to a pharmaceutical compositioncomprising as active ingredient a therapeutically effective amount of atleast one or more ASCT1 inhibitor and/or at least one or more ASCT2inhibitor and a pharmaceutically acceptable adjuvant, diluents orcarrier.

In another aspect the invention relates to a method for the enhancementof visual function, the method comprising administering to a subject inneed thereof an ophthalmically acceptable pharmaceutical compositioncontaining a therapeutically effective amount of one or more D-serinetransporter inhibitor compounds.

In another aspect the invention relates to a method for the enhancementof visual function, the method comprising administering to a subject inneed thereof an ophthalmically acceptable pharmaceutical compositioncontaining a therapeutically effective amount of one or more D-serinetransporter inhibitor compounds selected from the group consisting ofthe Glycine/Alanine family, the Glutamine/Asparagine family, theTryptophan Family, the Phenylglycine family, the Phenylalanine family,the Cysteine family, the Serine/Threonine family, the Cyclic Amino Acidfamily and the Proline family.

In another aspect the invention relates to a method for the enhancementof visual function, the method comprising administering to a subject inneed thereof an ophthalmically acceptable pharmaceutical compositioncontaining a therapeutically effective amount of one or more D-serinetransporter inhibitor compounds selected from the group consisting ofL-gamma-glutamyl-4-nitroanilide, L-4-hydroxyphenylglycine,L-4-fluorophenylglycine, L-phenylglycine, trans-4-hydroxy-L-proline andR-gamma-2,4-dichlorobenzyl-L-proline.

In another aspect the invention relates to a method for the enhancementof visual function, the method comprising administering to a subject inneed thereof an ophthalmically acceptable pharmaceutical compositioncontaining a therapeutically effective amount of at least one or moreASCT1 inhibitor and/or at least one or more ASCT2 inhibitor compounds.

In another aspect the invention relates to a pharmaceutical compositioncomprising as active ingredient a therapeutically effective amount ofL-gamma-glutamyl-4-nitroanilide and a pharmaceutically acceptableadjuvant, diluents or carrier.

In another aspect the invention relates to a pharmaceutical compositioncomprising as active ingredient a therapeutically effective amount ofL-4-hydroxyphenylglycine and a pharmaceutically acceptable adjuvant,diluents or carrier.

In another aspect the invention relates to a pharmaceutical compositioncomprising as active ingredient a therapeutically effective amount ofL-4-fluorophenylglycine and a pharmaceutically acceptable adjuvant,diluents or carrier.

In another aspect the invention relates to a pharmaceutical compositioncomprising as active ingredient a therapeutically effective amount ofL-phenylglycine and a pharmaceutically acceptable adjuvant, diluents orcarrier.

In another aspect the invention relates to a pharmaceutical compositioncomprising as active ingredient a therapeutically effective amount oftrans-4-hydroxy-L-proline and a pharmaceutically acceptable adjuvant,diluents or carrier.

In another aspect the invention relates to a pharmaceutical compositioncomprising as active ingredient a therapeutically effective amount ofR-gamma-2,4-dichlorobenzyl-L-proline and a pharmaceutically acceptableadjuvant, diluents or carrier.

In another aspect, the present invention relates to pharmaceuticalcompositions comprising D-serine transporter inhibitors and therapeuticmethods using such pharmaceutical compositions in methods for thetreatment of visual system disorders.

In another aspect, the present invention relates to a method for theenhancement of visual function comprising administration of one or moreD-serine transporter inhibitors by different administration routes.D-serine transporter inhibitors were identified as compounds thatinhibit transport mechanisms in neurons and astrocytes, in D-serinetransport assays in vitro.

Enhancement of visual function means administering one or more of theD-serine transport inhibitor compounds to improve the visual function,to alleviate its severity, to prevent the onset of a disorder, and toprevent its reoccurrence. Visual function includes visual acuity, visualfield, night vision, color vision, dark/light adaptation, contrastsensitivity, binocular vision, motion detection, etc.

In another aspect the present invention relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone D-serine transporter inhibitor compound, said compound being presentalone or in combination with one or more pharmaceutically acceptableexcipients.

In another aspect the present invention relates to a method for thetreatment of visual system disorders caused by a deficit inN-methyl-D-aspartate receptor function, the method comprisingadministering to a subject in need thereof an ophthalmically acceptablepharmaceutical composition containing a therapeutically effective amountof one or more D-serine transporter inhibitor compounds.

In another aspect the present invention relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone compound selected from the group consisting of ASCT1 inhibitor,ASCT2 inhibitor, and combinations thereof, said compound being presentalone or in combination with one or more pharmaceutically acceptableexcipients.

In another aspect the present invention relates to a method for thetreatment of visual system disorders caused by a deficit inN-methyl-D-aspartate receptor function, the method comprisingadministering to a subject in need thereof an ophthalmically acceptablepharmaceutical composition containing a therapeutically effective amountof one or more compounds selected from the group consisting of ASCT1inhibitor, ASCT2 inhibitor, and combinations thereof.

In another aspect the present invention relates to a method for theenhancement of visual function, the method comprising administering to asubject in need thereof an ophthalmically acceptable pharmaceuticalcomposition containing a therapeutically effective amount of one or morecompounds selected from the group consisting of ASCT1 inhibitor, ASCT2inhibitor, and combinations thereof.

In another aspect the present invention relates to a method for thetreatment of visual system disorders caused by a deficit inN-methyl-D-aspartate receptor function, the method comprisingadministering to a subject in need thereof an ophthalmically acceptablepharmaceutical composition containing a therapeutically effective amountof L-gamma-glutamyl-4-nitroanilide L-4-hydroxyphenylglycine,L-4-fluorophenylglycine, L-phenylglycine, trans-4-hydroxy-L-proline,R-gamma-2,4-dichlorobenzyl-L-proline.

The actual amount of the compound to be administered in any given casewill be determined by a physician taking into account the relevantcircumstances, such as the severity of the condition, the age and weightof the patient, the patient's general physical condition, the cause ofthe condition, and the route of administration.

The patient will be administered the compound orally in any acceptableform, such as a tablet, liquid, capsule, powder and the like, or otherroutes may be desirable or necessary, particularly if the patientsuffers from nausea. Such other routes may include, without exception,transdermal, parenteral, subcutaneous, intranasal, via an implant stent,intrathecal, intravitreal, topical to the eye, back of the eye, front ofthe eye, intramuscular, intravenous, and intrarectal modes of delivery.Additionally, the formulations may be designed to delay release of theactive compound over a given period of time, or to carefully control theamount of drug released at a given time during the course of therapy.

In another embodiment of the invention, there are providedpharmaceutical compositions including at least one compound of theinvention in a pharmaceutically acceptable carrier thereof. The phrase“pharmaceutically acceptable” means the carrier, diluent or excipientmust be compatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

Pharmaceutical compositions of the present invention can be used in theform of a solid, a solution, an emulsion, a dispersion, a patch, amicelle, a liposome, and the like, wherein the resulting compositioncontains one or more compounds of the present invention, as an activeingredient, in admixture with an organic or inorganic carrier orexcipient suitable for enteral or parenteral applications. Inventioncompounds may be combined, for example, with the usual non-toxic,pharmaceutically acceptable carriers for tablets, pellets, capsules,suppositories, solutions, emulsions, suspensions, and any other formsuitable for use. The carriers which can be used include glucose,lactose, gum acacia, gelatin, mannitol, starch paste, magnesiumtrisilicate, talc, corn starch, keratin, colloidal silica, potatostarch, urea, medium chain length triglycerides, dextrans, and othercarriers suitable for use in manufacturing preparations, in solid,semisolid, or liquid form. In addition auxiliary, stabilizing,thickening and coloring agents and perfumes may be used. Inventioncompounds are included in the pharmaceutical composition in an amountsufficient to produce the desired effect upon the process or diseasecondition.

Pharmaceutical compositions containing invention compounds may be in aform suitable for oral use, for example, as tablets, troches, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsions,hard or soft capsules, or syrups or elixirs. Compositions intended fororal use may be prepared according to any method known in the art forthe manufacture of pharmaceutical compositions and such compositions maycontain one or more agents selected from the group consisting of asweetening agent such as sucrose, lactose, or saccharin, flavoringagents such as peppermint, oil of wintergreen or cherry, coloring agentsand preserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets containing invention compounds inadmixture with non-toxic pharmaceutically acceptable excipients may alsobe manufactured by known methods. The excipients used may be, forexample, (1) inert diluents such as calcium carbonate, lactose, calciumphosphate or sodium phosphate; (2) granulating and disintegrating agentssuch as corn starch, potato starch or alginic acid; (3) binding agentssuch as gum tragacanth, corn starch, gelatin or acacia, and (4)lubricating agents such as magnesium stearate, stearic acid or talc. Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed.

In some cases, formulations for oral use may be in the form of hardgelatin capsules wherein the invention compounds are mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin. They may also be in the form of soft gelatin capsules whereinthe invention compounds are mixed with water or an oil medium, forexample, peanut oil, liquid paraffin or olive oil.

The pharmaceutical compositions may be in the form of a sterileinjectable suspension. This suspension may be formulated according toknown methods using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example, as a solution in 1,3-butanediol.Sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides, fatty acids (including oleicacid), naturally occurring vegetable oils like sesame oil, coconut oil,peanut oil, cottonseed oil, etc., or synthetic fatty vehicles like ethyloleate or the like. Buffers, preservatives, antioxidants, and the likecan be incorporated as required.

Invention compounds may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionsmay be prepared by mixing the invention compounds with a suitablenon-irritating excipient, such as cocoa butter, synthetic glycerideesters of polyethylene glycols, which are solid at ordinarytemperatures, but liquefy and/or dissolve in the rectal cavity torelease the drug.

Since individual subjects may present a wide variation in severity ofsymptoms and each drug has its unique therapeutic characteristics, theprecise mode of administration and dosage employed for each subject isleft to the discretion of the practitioner.

An opthalmically acceptable pharmaceutical composition is one that canbe administered topically to the eye of a subject in need thereof.Comfort to the subject being administered the composition should bemaximized, but other considerations, such as drug stability, maynecessitate a pharmaceutical composition that provides less than optimalcomfort. In such a case, the composition should be formulated such thatit is tolerable to a subject being administered the compositiontopically.

The claimed pharmaceutical composition can be administered topically inthe form of solutions or suspensions, ointments, gels, creams, etc. A“pharmaceutically acceptable excipient” is one that is compatible withthe active ingredient of the composition and not harmful to the subjectbeing administered the pharmaceutical composition. Solutions forophthalmic application are often prepared using physiological saline asa major vehicle. Other vehicles include polyvinyl alcohol, povidone,hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose,hydroxyethyl cellulose, and purified water. Examples of usefulexcipients also include preservatives, buffers, other pH adjustors,tonicity adjustors, surfactants, antioxidants, and chelating agents.

Useful preservatives include benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate and phenylmercuric nitrate. Examplesof buffers include phosphate, borate, sulfate, acetate, and citratebuffers. Acids or bases may be used to adjust the pH of the compositionsas needed. Examples of tonicity agents include glycerin, mannitol,sodium chloride and potassium chloride. Useful surfactants include, forexample, Tween 80. Examples of ophthalmically acceptable antioxidantsinclude sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene. A usefulchelating agent is edentate disodium.

Mixtures of two or more of any suitable excipients may be used.

Aside from topical application to treat diseases affecting the eyeincluding glaucoma, pharmaceutical compositions containing at least onecompound of formula (I) can also be administered periocularly,intraocularly, or by other effective means available in the art.

Persons skilled in the art would readily understand that a drugcontaining one or more of the compounds disclosed herein can beconfected as a powder, pill, tablet or the like, or as a solution,emulsion, suspension, aerosol, syrup or elixir suitable for oral orparenteral administration or inhalation. For solid dosage forms ormedicaments, non-toxic solid excipients for admixture with compoundsdisclosed herein include, but are not limited to, pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharin,polyalkylene glycols, talcum, cellulose, glucose, sucrose, and magnesiumcarbonate. The solid dosage forms may be coated by a material such asglyceryl monostearate or glyceryl distearate, which is utilized in knowntechniques to delay disintegration and absorption in thegastrointestinal tract for the purpose of providing a sustained actionover a longer period. Solid dosage forms may also be coated by thetechniques described in U.S. Pat. Nos. 4,256,108, 4,166,452 and4,265,874 to form osmotic therapeutic tablets for control release.

Pharmaceutically administrable liquid dosage forms can, for example,comprise a solution or suspension of at least one of the compoundsdisclosed herein and optional pharmaceutical adjutants in a carrier,such as water, saline, aqueous dextrose, glycerol, ethanol and the like.The liquid dosage forms may also contain nontoxic auxiliary substancessuch as wetting or emulsifying agents, pH buffering agents and the like.Examples of such auxiliary agents include sodium acetate, sorbitanmonolaurate, triethanolamine, sodium acetate, triethanolamine oleate,etc. Methods for preparing such dosage forms are well-known to personsskilled in the art (see, for example, Reminton's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 16^(th) Edition, 1980).

Parenteral administration is generally characterized by subcutaneous,intramuscular, or intravenous injection. Injectables can be prepared asliquid solutions or suspensions, solid forms that can be reconstitutedinto solutions or suspensions prior to injection, or as emulsions.Suitable excipients include water, saline dextrose, glycerol, ethanoland the like. Such injectable pharmaceutical compositions may alsocontain minor amounts of non-toxic auxiliary substances such as wettingor emulsifying agents, pH buffers and the like.

Examples mentioned herein are not intended to limit the scope of theinvention in any way.

Using D-serine transport assays in vitro, we have identified compoundsthat inhibit transport mechanisms in neurons and astrocytes. In Table 1,the activities of amino acid analogs to inhibit D-serine transport areshown. Under the assay conditions used, the sodium-independent transportof D-serine by rodent forebrain synaptosomes is mediated by asc-1(Rutter et al., 2007), and the sodium-dependent transport of D-serineinto astrocytes in culture is mediated by an ASCT transporter, ASCT2according to the literature (Ribeiro et al., 2002).

The data from Table 1 shows that transport of D-serine into neurons andinto astrocytes can be pharmacologically distinguished. Analogs ofglutamine, phenylglycine, asparagine, cysteine and proline were able toselect between the two transport systems.

To determine the effects of transport inhibition on NMDA receptorfunction, compounds were tested for their ability to affect NMDAreceptor-mediated synaptic responses in brain slice preparations.L-4-hydroxyphenylglycine (L-4OHPG) potentiated NMDA receptor mediatedexcitatory post-synaptic currents (EPSC's) in the CA1 region of thehippocampus (FIG. 1A and FIG. 1B). In the visual cortex slice, LTPevoked by theta burst stimulation was enhanced by L-4OHPG. L-4OHPGenhanced LTP in a concentration-dependent manner, and its effects werecompletely prevented by inclusion of D-AAO in the perfusion medium,indicating that its ability to enhance synaptic plasticity was dependenton extracellular D-serine (FIG. 2B). Importantly, none of the compoundsidentified as D-serine transport inhibitors had significant directeffects on the NMDA receptor (or other glutamate receptor sub-types) asassessed in cultured hippocampal neurons.

In an attempt to understand the relative contributions of the neuronaland astrocyte D-serine transporters to the observed ability of compoundsto enhance NMDA receptor-mediated responses, correlations were madebetween the effects of compounds in the transport assays and in thevisual cortex slice LTP assay. A poor correlation was found between theeffects in the neuronal transport assay and LTP (FIG. 3A, r²=0.047)however an excellent correlation existed between the effects in theastrocyte transport assay and LTP (FIG. 3B, r²=0.902). This indicatedthat the transporters present in astrocytes are those that regulateextracellular D-serine to influence NMDA receptor-mediated synapticevents.

The sodium-dependent D-serine transporter in astrocytes has beenreported to be ASCT2 (Ribeiro et al., 2002). In the D-serine transportexperiments in astrocytes, we noticed that some compounds producedinhibition curves that exhibited two components, suggesting that morethan one transport component was present. In particular, two compoundsdefined the two components. L-glutamine showed higher affinity for acomponent that represented approximately 40% of the D-serine transport,and L-trans-4-hydroxyproline (L-t-4OHPro) showed higher affinity for acomponent that represented approximately 60% of the D-serine transport(FIG. 4). Competition studies with each of these compounds in thepresence of the other indicated that L-glutamine had high affinity forthe component with low affinity for L-t-4OHPro and vice versa. PCRstudies have indicated that both ASCT1 and ASCT2 transporter sub-typesare present in astrocytes (Yamamoto et al., 2004). However, functionalexpression of ASCT1 and ASCT2 in heterologous systems has indicatedthat, unlike ASCT2, ASCT1 does not transport D-serine (Shafqat et al.,1993). L-glutamine is reported to have high affinity for ASCT2 (range of23-70 μM; Utsunomiya-Tate et al., 1996; Broer et al., 1999;Torres-Zamorano et al., 1998), and one report indicates that L-t-4OHProhas high affinity for ASCT1 (Pinilla-Tenas et al., 2003). We confirmedthe selectivity of L-glutamine and L-t-4OHPro for the ASCT sub-types byexamining transport in HEK cells heterologously expressing human ASCT1and ASCT2. For these experiments, [³H]L-serine was used since it is ahigh affinity substrate for both sub-types. As shown in FIG. 5,L-glutamine inhibited transport and showed selectivity towards ASCT2,whereas L-t-4OHPro showed selectivity towards ASCT1. Consequently, thetwo components of transport observed in astrocytes most likely representASCT1 (L-t-4OHPro-preferring) and ASCT2 (L-glutamine-preferring). Ifthis is the case, however, it would suggest that ASCT1 does indeedtransport D-serine, contrary to the literature report (Pinilla-Tenas etal., 2003). To investigate this, we examined transport intoASCT1-expressing HEK cells. As shown in FIG. 6A, [³H]D-serine wastransported into ASCT1-expressing HEK cells in a sodium-dependent mannerand to a similar degree to the transport observed in ASCT2-expressingHEK cells. In addition, [³H]L-serine transport was completely inhibitedby D-serine in astrocytes and ASCT1 and ASCT2-expressing HEK cell lines(FIG. 6B) as would be expected if D-serine interacts with bothtransporter sub-types. Consequently, we have discovered that D-serine isindeed a substrate for ASCT1 with an affinity similar to that for ASCT2.

Given this evidence that transport into astrocytes is mediated by bothASCT1 and ASCT2, which of these transporter sub-types is primarilyresponsible for the inhibition of D-serine transport that leads to theenhancement of LTP observed? To address this, we examined the ability ofthe inhibitors identified in the astrocyte transport assay and thatenhance LTP to inhibit transport in the HEK cells expressing each ASCTsub-type. As shown in Table 2, L-glutamine and the L-glutamine analogL-gamma-glutamyl-4-nitroanilide (L-GPNA) were selective for ASCT2.L-trans-4OHPro was selective for ASCT1. The phenylglycine analogs,isomers of serine, asparagine and cyclopropylglycine showed equalability to inhibit both sub-types. Correlations between the IC₅₀ valuesfor transport inhibition at the sub-types and the thresholdconcentrations to enhance LTP revealed no significant correlation withASCT1 (FIG. 7 a) but a significant correlation with ASCT2 (FIG. 7 b),however the best correlation was obtained when the contribution of bothsub-types was taken into account (product of the IC₅₀'s for both ASCT1and ASCT2; FIG. 7 c). This suggests that both sub-types are importantfor the enhancement of LTP and that dual sub-type inhibitors are themost effective compounds.

Examples of D-Serine Transporter Inhibitors

It has been found that certain amino acids of the Glycine/Alaninefamily, the Glutamine/Asparagine family, the Tryptophan Family, thePhenylglycine family, the Phenylalanine family, the Cysteine family, theSerine/Threonine family, the Cyclic Amino Acid family and the Prolinefamily are examples of D-serine transporter inhibitors.

The following are non-limiting examples of D-serine transporterinhibitors which are useful in the practice of the present invention.The amino acids that were tested for D-serine transport inhibitionproperties were obtained from Sigma-Aldrich, Tocris Bioscience, TygerChemical Scientific, Bachem, ChemBridge Corporation, Matrix Scientific,PI Chemicals Inc., Toronto Research Chemicals and Maybridge Chemicals.

Table of Active Compounds by Amino Acid Family Criterion for activity:≧25% inhibition of [³H]D-serine transport into rat hippocampalastrocytes at 1 mM Compound Isomer Glycine/Alanine Family glycinealanine L 2-aminobutyrate L 2-allylglycine DL valine L3-(methylamino)alanine L 1-aminocyclopropane-1-carboxylic acid1-aminocyclobutane-1-carboxylic acid 1-aminocyclopentane-1-carboxylicacid α-cyclopropylglycine L phenylglycine L tetrazol-5yl glycine DL3-thienylglycine L aminocyclohexyl acetic acid L aminofuran-2-yl aceticacid L amino-(2,3-dihydro-benzo[1,4]dioxin-6-yl)-acetic DL acidaminonaphthalen-1-yl acetic acid L aminobicyclo[2.2.1]hept-5-en-2-ylacetic acid DL dihydrophenylglycine D 1-adamantyl(amino)acetic acid2-aminoadamantine-2-carboxylic acid 3-benzoylalanine DL3-(2-thienyl)-alanine L 3-cyclopentyl-alanine L 3(2-naphthyl)-alanine L3-benzothienylalanine L azidohomoalanine L homopropargylglycine L valineL norvaline L alanine D Glutamine/Asparagine Family glutamine Lglutamate-γ-hydroxamate L glutamate-γ-4-nitroanilide Lglutamate-γ-anilide DL glutamate-γ-(α-naphthylamide) Lglutamate-γ-(β-naphthylamide) L glutamate-γ-(β-naphthylamide) Lglutamate-γ-methylester L glutamate-γ-ethylester L asparagine Lasparagine D N-4-phenylasparagine DL kynurenine L kynurenine D 3-hydroxykynurenine DL 2-amino-succinic acid 4-ethylester DL aspartate benzylester L 6-diazo-5-oxo-norleucine L Tryptophan Family tryptophan L6-fluorotryptophan DL 5-fluorotryptophan L 4-fluorotryptophan DL5-hydroxytryptophan L Phenylglycine Family phenylglycine L4-hydroxyphenylglycine L 4-fluorophenylglycine L 4-methoxyphenylglycineDL amino-(4-nitro-phenyl)-acetic acid DL 4-trifluoromethylphenylglycineL 3-hydroxyphenylglycine L amino-(3-fluoro-phenyl)-acetic acid DLamino-(3-bromo-phenyl)-acetic acid DL 3-trifluoromethylphenylglycine DLamino-(3-nitro-phenyl)-acetic acid DL 2-fluorophenylglycine DLamino-o-tolyl-acetic acid L 2-chlorophenylglycine DL3,4-difluorophenylglycine DL 3-chloro-4-fluorophenylglycine DL3-fluoro-4-methylphenylglycine DL 4-fluoro-3-methylphenylglycine DL3-carboxy-4-hydroxyphenylglycine L 2-Cl, 5-OH phenylglycine DL3,4-dihydroxyphenylglycine DL 3,5-dihydroxyphenylglycine DL4-carboxy-3-hydroxyphenylglycine DL 2-phenylglycine methylester L(4-methoxyphenyl)(methylamino)acetic acid DL 2-hydroxyphenylglycine DLamino-(2,3-dihydrobenzo [1,4]dioxin-6-yl) acetic acid DLamino-benzo[1,3]dioxo1-5-yl acetic acid DL2-amino-2-[3-hydroxy-4-(hydroxymethyl)phenyl]acetic acid DL(4-fluorophenyl)-morpholin-4yl-acetic acid DL cyclopropylalanine LPhenylalanine Family homophenylalanine L 2-amino-5-phenylpentanoic acidL 4-hydroxyphenylalanine L 3,4-dihydroxyphenylalanine L Quisqualic acidL Cysteine Family cysteine L S-methyl-cysteine L S-ethyl-cysteine LS-phenyl-cysteine L S-benzyl-cysteine L S-(4-methylphenyl)-cysteine Lpenicillamine L homocysteine L Serine/Threonine Family serine L serine Dthreonine L threonine D threonine L-allo threonine DL-alloO-methylserine DL O-acetylserine L benzylserine L beta (2-thienyl)serineDL 3-pyridylserine DL serine methylester L serine-beta-naphthylamide Lmethionine L 4-hydroxy-isoleucine L homoserine D homoserine L CyclicAmino Acid Family 1-amino-1-carboxycyclopropane1-amino-1-carboxycyclobutane 1-amino-1-carboxycyclopentane homocysteinethiolactone L homoserine lactone L Proline Family proline L3,4-dehydroproline L 4-hydroxy-L-proline trans 4-fluoro-L-proline trans4-fluoro-L-proline cis γ-benzyl-L-proline R γ-(4-fluorobenzyl)-L-prolineR 1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid S2,3,4,9-tetrahydro-1H-beta-carboline-3-carboxylic DL acid2,3-dihydro-1H-isoindole-1-carboxylic acid DL4H-thieno[3,2-b]pyrrole-5-carboxylic acid azetidine-2-carboxylic acid Lproline-beta naphthylamide L trans-4-cyclohexylproline Ltrans-4-hydroxyproline-naphthylamide L4,6-Dichloro-3-[(1E)-3-oxo-3-(phenylamino)-1-propenyl]-1H-indole-2-carboxylic acid(2S,3S,4S)-Carboxy-4-(1-methylethenyl)-3- pyrrolidineacetic acid4-methoxy-7-nitro-1H-indolinyl amide (E)-4,6-Dichloro-3-(2-phenyl-2-carboxyethenyl)indole-2-carboxylic acid γ-allyl-L-proline Raziridine-2-carboxylic acid L γ-(4-nitrobenzyl)-L-proline Rtrans-4-phenylproline L γ-(3,4-difluorobenzyl)-L-proline Rγ-(3-thienylmethyl)-L-proline R γ-(4-methylbenzyl)-L-proline Rγ-(2-naphthylenylmethyl)-L-proline R γ-propynyl-L-proline Rγ-(3-fluorobenzyl)-L-proline R γ-(2-fluorobenzyl)-L-proline Rγ-(4-bromobenzyl)-L-proline R γ-(4-chlorobenzyl)-L-proline HCl Rγ-(4-iodobenzyl)-L-proline HCl R 4H-thieno[3,2-b]pyrrole-5-carboxylicacid γ-(2-trifluromethylbenzyl)-L-proline HCl Rγ-(4-tertbutylbenzyl)-L-proline HCl R 3-phenylprolineγ-(2-cyanobenzyl)-L-proline HCl R γ-(2-methylbenzyl)-L-proline HCl Rγ-(3-trifluoromethyl-benzyl)-L-proline HCl Rγ-(3-phenyl-allyl)-L-proline HCl (Boc?) Rγ-(1-naphthalenylmethyl)-L-proline HCl R4-(3-chlorobenzyl)pyrrolidine-2-carboxylic acid HCl 2S, 4S4-(3-chlorobenzyl)pyrrolidine-2-carboxylic acid HCl 2S, 4R4-benzyl-L-proline S γ-(2-furanylmethyl)-L-proline Sγ-(3-chlorobenzyl)-L-proline HCl R γ-(2-pyridinylmethyl)-L-proline 2HClS 4-(3-chlorophenoxy)pyrrolidine-2-carboxylic acid 2S, 4R HCl4-(3-chlorophenoxy)pyrrolidine-2-carboxylic acid 2S, 4S HClγ-(2-iodobenzyl)-L-proline HCl R γ-(3-benzothienylmethyl)-L-proline HClR γ-(2-bromobenzyl)-L-proline HCl R γ-(4-trifuoromethylbenzyl)-L-prolineHCl R γ-(3-bromobenzyl)-L-proline HCl R γ-(4-pyridinylmethyl)-L-prolineHCl R γ-(4-cyanobenzyl)-L-proline HCl R γ-(3-cyanobenzyl)-L-proline HClR γ-(3,4-dichlorobenzyl)-L-proline HCl R γ-(2-chlorobenzyl)-L-prolineHCl R γ-(2,4-dichlorobenzyl)-L-proline HCl R γ-propynyl-L-proline HCl Rγ-(2-cyanobenzyl)-L-proline HCl R 3-methyl-2-pyrrolidine-2-carboxylicacid 2S, 3S 3-phenyl-2-pyrrolidine-2-carboxylic acid 2S, 3R(E)-4,6-Dichloro-3-(2-phenyl-2- carboxyethenyl)indole-2-carboxylic acid4,6-Dichloro-3-[(1E)-3-oxo-3-(phenylamino)-1-propenyl]-1H-indole-2-carboxylic acidCarboxy-4-(1-methylethenyl)-3-pyrrolidineacetic (2S, 3S, 4S) acid4-methoxy-7-nitro-1H-indolinyl amide

The compounds identified here in assays of D-serine transport areinhibitors of the transporter sub-types ASCT1 (SLC1A4) and ASCT2(SLC1A5), as confirmed is transport assays using HEK cells thatheterologously express human ASCT1 or ASCT2. This includes the compoundsL-gamma-glutamyl-4-nitroanilide, L-4-hydroxyphenylglycine,L-4-fluorophenylglycine, L-phenylglycine, trans-4-hydroxy-L-proline andR-gamma-2,4-dichlorobenzyl-L-proline. These compounds have IC₅₀ valuesless than 2 mM in one or both assays of transport in HEK cellsexpressing ASCT1 or ASCT2.

To investigate the ability of compounds to improve visual function invivo, compounds identified in the transport and in vitro LTP assays wereselected for study in rodent models. These experiments show that L-4OHPGand L-4FPG enhance visual function in normal rats as assessed by sweepVEP (FIGS. 8 and 9), and that L-4OHPG enhances visually-evoked signalsfrom the visual cortex in rats with optic nerve crush (FIG. 10). L-4OHPGalso enhanced sweep VEP in normal rabbits (FIG. 11). In a model ofmacular degeneration, rats with damaged retinas following blue-lighttreatment were found to have a deficit in contrast sensitivity.Treatment with L-4OHPG showed a significant improvement in contrastsensitivity, restoring it towards normal levels (FIG. 12). Thus, we haveshown that compounds which inhibit D-serine transport can improve visualperformance in normal rats and rabbits and in a two retinal diseasemodels where visual performance has been impaired.

General Procedures Followed in Obtaining Experimental Data

Electrophysiological Recording from Rat Hippocampal Slices (FIGS.1A-1B):

350 μM thick hippocampal slices were prepared from 21- to 35-year-oldrats using Leica VT1000S-microtome. Slices were perfused with ACSFcontaining: 121 mM NaCl, 2.5 mM KCl, 2.0 mM Mg₂SO₄, 2.0 CaCl₂, 1 mMNaH₂PO₄, 26.2 NaHCO₃, and 11 mM glucose, which was equilibrated with 5%CO₂/95% O₂. Experiments were performed in a recording chamber on thestage of an Olympus BX-61wi microscope with infrared DIC optics forvisualizing whole-cell patch-clamp recordings. EPSPs were recorded fromCA1 pyramidal neurons by stimulating the Schaffer collateral-commissuralpathway using a bipolar tungsten electrode. The recording pipettes werefilled with regular ICM containing: 120 mM Cs-gluconate, 5 mM NaCl, 10mM KCl, 0.1 mM CaCl₂, 1 mM EGTA, 2 mM MgCl₂, 10 mM HEPES, 2 mM Na-ATP, 2mM Na₂-phosphocreatine, and 0.25 mM Na-GTP, pH 7.3 (290 mOsm).

To measure NMDA-mediated EPSCs, extracellular Mg₂SO₄ was lowered to 0.2mM and 2 μM NBQX and 100 μM picrotoxin were added in the ACSF. 1 μM7-CKY (7-chlorokynurenic acid) was added to improve the sensitivity ofEPSC_(NMDA) to D-serine.

Long Term Potentiation in Primary Visual Cortex (FIG. 2)

Long Term Potentiation (LTP) in primary visual cortex has been used as acellular model for visual cortex plasticity and has functionalconsequences on visual evoked responses. NMDA receptors play a criticalrole in visual cortex LTP induction.

Visual Cortex Slice Physiology:

Following decapitation of the rat, the brain was rapidly removed andimmersed in ice-cold artificial cerebrospinal fluid (ACSF) containing124 mM NaCl, 3 mM KCl, 1.25 mM KH₂PO₄, 3.4 mM CaCl₂, 2.5 mM MgSO₄, 26 mMNaHCO₃, and 10 mM D-glucose. A block of visual cortex was created byremoving the frontal ⅔ portion of the brain and the cerebellum. Coronalvisual cortex slices of 375 μm were prepared from adult Sprague Dawley(SD) rats using a vibratome (VT 1000 S; Leica). The slices weremaintained in an interface recording chamber perfused with preheatedACSF. Slices were continuously perfused with this solution at a rate of1.00-1.50 ml/min while the surface of the slices was exposed to warm,humidified 95% O₂/5% CO₂ and maintained at 31±1° C. Visual cortex sliceswere allowed to recover for 1 hr before recording began. A singlestimulating and recording electrode were placed in layer IV and III,respectively, to generate and record a field excitatory postsynapticpotentials (fEPSPs). Pulses were administered every 20 s using a currentthat produced a fEPSP that was 50% of the maximum spike free response.An input-output (IO) curve was done to determine the stimulation neededto achieve a stable baseline. Following a 15 min stable baselinerecording period, a train of 5 theta bursts (each burst containing fourpulses at 100 Hz with an inter-burst interval of 200 ms) were deliveredto the slice. This was repeated 2 additional times with a 1 minuteintertrain interval, and the level of LTP was recorded for at least 30min. Changes in amplitude of the synaptic response were used to measurethe extent of LTP because it was determined to be the more consistentparameter than the slope of the response. Control LTP values wereobtained from slices not treated with drug. Different slices were usedto study drug effects on LTP. After a 15 min baseline recording period,the compounds of interest were infused for 15 minutes followed by LTPinduction. Washout of the compounds began 5 minutes after tetanization.Recording of the amplitude before, during, and after drug infusion wasdone.

*DAAO Assay (FIG. 2B):

For experiments with DAAO, 0.2 unit/ml of DAAO were infused with orwithout the compounds of interest for 15 minutes before LTP induction.

*Sweep VEP (FIGS. 8-12):

Data gathered through the sweep visually evoked potential assessment(sweep VEP, sVEP) show that L-4FPG and L-4OHPG enhance visual functionin normal rats and rabbit and that L-4OHPG enhances remaining visualfunction in rats with optic nerve crush.

Sweep visually evoked potential (sweep VEP, sVEP), which was firstintroduced by Regan [1] in 1973, has become an important technique tomeasure visual function. It is an objective method that can be used toassess visual acuity (VA) and contrast sensitivity (CS) in infants,young children and people with special needs. It was adapted to measureVA and CS in animals.

VEP Recording in Rats:

The recording electrodes were permanently implanted into the rightvisual cortex of Long Evans rats at lambda and 4.5 mm lateral to themidline, to a depth of 800 microns (layer III/iV). A reference electrodewas placed epidurally on the midline 1.2 mm anterior to bregma. Allrecordings were conducted in awake rats starting at least two weeksafter recovery from surgery. During recording the rats were alert andrestrained in a home-made restrainer. They were habituated 2-3 timespre-surgery and at least three more times during seven dayspost-surgery. PowerDiva software from Anthony Norcia (Smith KettlewellInstitute of Visual Sciences) was used for data acquisition andanalysis. Similar recording was performed in rabbits (FIG. 11) exceptthe screw electrodes were place on top of the skull.

Visual Stimuli for Visual Acuity Measurement:

Stimuli were presented on a CRT computer monitor and consist offull-field sine-wave gratings at 80% contrast, reversing at 6.25 Hz.VEPs were elicited by horizontally oriented gratings. The display waspositioned 24 cm in front of the rat and centered at the verticalmeridian. Mean luminance was held constant at 20 cd. For sVEP in normalrats (slides 76-79), one stimulus presentation (one trial) consists of aspatial frequency sweep decreasing from 1.6 to 0.03 cycles/degree in 15linear steps. A total of 20 trials were collected. Visual acuity (VA)thresholds were estimated using PowerDiva software. For fixed frequencystimulus in optic nerve crushed (ONC) rats (slides 80-83), the spatialfrequency was fixed at 0.2 or 0.5 cycles/degree. Each trial lasts for 15s. A total of 5 trials were collected and the signal powers werecalculated.

Visual Stimuli for Contrast Sensitivity Measurement:

One stimulus presentation (one trial) consists of a contrast sweepincreasing from 2.5 to 70% in 15 log steps. A total of 20 to 30 trialswere collected. Contrast thresholds (CT) were estimated using PowerDivasoftware. Contrast sensitivity (CS) is calculated as 1/CT.

Blue-light treatment damages photoreceptors in the retina, and has beenproposed as a model of age related macular degeneration (ARMD; Wielguset al., 2010). In blue-light treated Long-Evans rats, contrastsensitivity, an important measure of visual performance, wassignificantly impaired.

Transport Experiments (Tables 1 and 2; FIGS. 4-6)

Cell-based assays: the transport of [³H]L- or D-serine was measured inprimary cultures of rat hippocampal astrocytes or in human embryonickidney (HEK) cells expressing ASCT transporter sub-types. For theastrocyte assays, cells were plated on either 24- or 96-well plates at adensity of 50,000 cells per well. For the HEK assays, cells were platedon coated 96-well plates at a density of 80,000 cells/well. Assays wereconducted in duplicate at room temperature in assay buffer consistingof: NaCl: 150 mM, KCl: 2 mM; MgCl₂: 1 mM; CaCl₂: 1 mM; HEPES: Trisbuffer: 10 mM, pH7.4. To assess the sodium-dependence of transport, NaClwas replaced in the assay buffer by equimolar choline chloride.Following aspiration of growth medium and 2 washes with assay buffer,cells were incubated with [³H]L- or D-serine at a final concentration of1 μM for 5 min (astrocytes) or 1 min (HEK cells), after which theincubation medium was aspirated and the cells washed twice with ice-coldassay buffer. Cells containing radiolabel were solubilized in 100 μl of1% Triton-X100 and an aliquot counted in a beta counter. IC₅₀ valueswere determined over a range of at least 6 concentrations and derivedfrom curve-fitting algorithms available in Graph Pad Prism 4.

Synaptosome assays: a P2 fraction of rat forebrain was prepared andassayed immediately. Aliquots of the P2 preparation (approx. 1 mg oforiginal tissue weight) were incubated in sodium-free assay buffer(CholineCl: 128 mM, KCl: 3.5 mM; KH₂PO₄: 1.5 mM; MgCl₂: 1 mM; CaCl₂: 1mM; glucose: 10 mM; Tris-acetate buffer: 10 mM, pH7.4) containing[³H]D-serine (final concentration of 50 nM) and test compounds induplicate for 4 mins at room temperature. The synaptosomes containingradiolabel were collected by filtration onto Whatman GF/C filters, andwashed twice with ice cold assay buffer. Filters were solubilized inscintillation fluid and radioactivity determined in a beta counter. IC₅₀values were determined as described for the cell-based assays above.

What is claimed is:
 1. A method for the treatment of visual systemdisorders caused by a deficit in N-methyl-D-aspartate receptor function,the method comprising administering to a subject in need thereof anophthalmically acceptable pharmaceutical composition containing atherapeutically effective amount of one or more D-serine transporterinhibitor compounds.
 2. The method for the treatment of visual systemdisorders, according to claim 1, the method comprising administering toa subject in need thereof an ophthalmically acceptable pharmaceuticalcomposition containing a therapeutically effective amount of one or moreD-serine transporter inhibitor compounds selected from theGlycine/Alanine family, the Glutamine/Asparagine family, the TryptophanFamily, the Phenylglycine family, the Phenylalanine family, the Cysteinefamily, the Serine/Threonine family, the Cyclic Amino Acid family andthe Proline family.
 3. The method for the treatment of visual systemdisorders, according to claim 2, the method comprising administering toa subject in need thereof an ophthalmically acceptable pharmaceuticalcomposition containing a therapeutically effective amount of one or moreD-serine transporter inhibitor compounds selected fromL-gamma-glutamyl-4-nitroanilide, L-4-hydroxyphenylglycine,L-4-fluorophenylglycine, L-phenylglycine, trans-4-hydroxy-L-proline andR-gamma-2,4-dichlorobenzyl-L-proline.
 4. The method for the treatment ofvisual system disorders, according to claim 3, the method comprisingadministering to a subject in need thereof an ophthalmically acceptablepharmaceutical composition containing a therapeutically effective amountof L-gamma-glutamyl-4-nitroanilide.
 5. The method for the treatment ofvisual system disorders, according to claim 3, the method comprisingadministering to a subject in need thereof an ophthalmically acceptablepharmaceutical composition containing a therapeutically effective amountof L-4-hydroxyphenylglycine.
 6. The method for the treatment of visualsystem disorders, according to claim 3, the method comprisingadministering to a subject in need thereof an ophthalmically acceptablepharmaceutical composition containing a therapeutically effective amountof L-4-fluorophenylglycine.
 7. The method for the treatment of visualsystem disorders, according to claim 3, the method comprisingadministering to a subject in need thereof an ophthalmically acceptablepharmaceutical composition containing a therapeutically effective amountof L-phenylglycine.
 8. The method for the treatment of visual systemdisorders, according to claim 3, the method comprising administering toa subject in need thereof an ophthalmically acceptable pharmaceuticalcomposition containing a therapeutically effective amount oftrans-4-hydroxy-L-proline.
 9. The method for the treatment of visualsystem disorders, according to claim 3, the method comprisingadministering to a subject in need thereof an ophthalmically acceptablepharmaceutical composition containing a therapeutically effective amountof R-gamma-2,4-dichlorobenzyl-L-proline.
 10. A pharmaceuticalcomposition comprising as active ingredient a therapeutically effectiveamount of at least one D-serine transporter inhibitor compound and apharmaceutically acceptable adjuvant, diluents or carrier.
 11. A methodfor the enhancement of visual function, the method comprisingadministering to a subject in need thereof an ophthalmically acceptablepharmaceutical composition containing a therapeutically effective amountof one or more D-serine transporter inhibitor compounds.
 12. The methodfor the enhancement of visual function, according to claim 11, themethod comprising administering to a subject in need thereof anophthalmically acceptable pharmaceutical composition containing atherapeutically effective amount of one or more D-serine transporterinhibitor compounds selected from the group consisting of theGlycine/Alanine family, the Glutamine/Asparagine family, the TryptophanFamily, the Phenylglycine family, the Phenylalanine family, the Cysteinefamily, the Serine/Threonine family, the Cyclic Amino Acid family andthe Proline family.
 13. The method for the enhancement of visualfunction, according to claim 12, the method comprising administering toa subject in need thereof an ophthalmically acceptable pharmaceuticalcomposition containing a therapeutically effective amount of one or moreD-serine transporter inhibitor compounds selected from the groupconsisting of L-gamma-glutamyl-4-nitroanilide, L-4-hydroxyphenylglycine,L-4-fluorophenylglycine, L-phenylglycine, and trans-4-hydroxy-L-proline.14. The method for the enhancement of visual function, according toclaim 13, the method comprising administering to a subject in needthereof an ophthalmically acceptable pharmaceutical compositioncontaining a therapeutically effective amount ofL-4-hydroxyphenylglycine.
 15. The method for the enhancement of visualfunction, according to claim 13, the method comprising administering toa subject in need thereof an ophthalmically acceptable pharmaceuticalcomposition containing a therapeutically effective amount ofL-4-fluorophenylglycine.
 16. The method for the enhancement of visualfunction, according to claim 13, the method comprising administering toa subject in need thereof an ophthalmically acceptable pharmaceuticalcomposition containing a therapeutically effective amount ofL-phenylglycine.
 17. The method for the enhancement of visual function,according to claim 13, the method comprising administering to a subjectin need thereof an ophthalmically acceptable pharmaceutical compositioncontaining a therapeutically effective amount ofL-gamma-glutamyl-4-nitroanilide.
 18. The method for the enhancement ofvisual function, according to claim 13, the method comprisingadministering to a subject in need thereof an ophthalmically acceptablepharmaceutical composition containing a therapeutically effective amountof trans-4-hydroxy-L-proline.
 19. The method for the enhancement ofvisual function, according to claim 13, the method comprisingadministering to a subject in need thereof an ophthalmically acceptablepharmaceutical composition containing a therapeutically effective amountof R-gamma-2,4-dichlorobenzyl-L-proline.
 20. The pharmaceuticalcomposition according to claim 10, wherein the D-serine inhibitorcompound is selected from: L-gamma-glutamyl-4-nitroanilide,L-4-hydroxyphenylglycine, L-4-fluorophenylglycine, L-phenylglycine,trans-4-hydroxy-L-proline, or R-gamma-2,4-dichlorobenzyl-L-proline.